Hexametra leidyi sp. n. (Nematoda: Ascarididae) from North American Pit Vipers (Reptilia: Viperidae)

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1 Proc. Helminthol. Soc. Wash. 51(1), 84, pp Hexametra leidyi sp. n. (Nematoda: Ascarididae) from North American Pit Vipers (Reptilia: Viperidae) DWIGHT D. BOWMAN Department of Parasitology, Tulane University, New Orleans, Louisiana ABSTRACT: Specimens of Hexametra from six canebrake rattlesnakes, Crotalus horridus atricaudatus, and one North American water moccasin, Agkistrodon piscivorus leucostoma, collected in Louisiana are described as Hexametra leidyi sp. n. Observations based on male and 21 female worms indicated that the specimens collected in Louisiana differed in several ways from the species reported from New World pit vipers. H. leidyi has lips significantly smaller (P < 0.001) than those of H. boddaertii from North American colubrid and viperid snakes. The dentigerous ridge of H. leidyi extends to the base of the lip and bears numerous small denticles, whereas in H. boddaertii the denticles are larger, fewer, and limited to the anterior margin of the lip. H. leidyi eggs are 90 ^m by 81 /um (mean), whereas those of H. boddaertii are 80 nm by 73 fj,m (mean). Also, the egg of H. leidyi has fewer pits on its surface than does the egg of H. boddaertii. H. leidyi eggshells have an average of 58 pits per 80 /urn, and in H. boddaertii the average is 90. Although previously synonymized with H. boddaertii sensu Sprent, 78, H. hexauterina (Skrjabin, ) from a Bothrops sp. in Paraguay was described as having denticles extending to the base of its lips and eggs similar in size to those of H. leidyi. However, as the type specimens of H. hexauterina could not be found, and its description appears inadequate, it is considered to be in a position of subjudice. Baird (60) described Ascaris boddaertii from a single female specimen collected from a West Indian colubrine snake, Mastigodryas (=Herpetodryas) boddaerti. Baylis (, ) redescribed this worm and placed the species in the genus Polydelphis Dujardin, 45. because the specimen had more than two uterine branches. Kreis (44) later transferred the species to the genus Hexametra that Travassos () had created for ascaridoids with six uterine branches. Until 78, when Sprent reviewed the genus Hexametra, the worm described by Baird was the only known specimen of H. boddaertii. Araujo (69) recovered worms from a Brazilian snake, Crotalus durrissus terrificus, and identified them as H. quadricornis (Wedl, 61). However, Sprent (78) concluded that the specimens examined by Araujo were H. boddaertii, not H. quadricornis. Sprent also placed two other species of Hexametra in synonymy with H. boddaertii. One of the species that Sprent considered as synonymous with H. boddaertii was Ascaris quadrangularis Schneider, 66. The type specimens of A. quadrangularis were collected from the same host and locality as the specimens examined by Araujo. However, Sprent found that the type collection was composed of a single Hexametra female mixed with several Ophidascaris specimens, and he felt that the female specimen was indistinguishable from H. boddaertii, which has taxonomic precedence. Sprent also indicated that H. hexauterina (Skrjabin, ) should be regarded as a synonym of H. boddaertii. The type specimens of H. hexauterina were collected from a snake, Bothrops sp., in Paraguay, and could not be found for examination. Although Sprent placed this species in synonymy with H. boddaertii, he did note that in Skrjabin's original description the denticles were shown to extend further posteriad on the lateral sides of the lips than they do in H. boddaertii. Also, Sprent (78) examined specimens of Hexametra from North American rattlesnakes in the collection of the U.S. National Parasite Collection and indicated that they were all specimens of H. boddaertii. He therefore concluded that all the specimens of Hexametra from the New World represented a single species H. boddaertii. Specimens collected recently from pit vipers in Louisiana were found to have characteristics that differentiate them from H. boddaertii sensu Sprent, 78, and they are described herein as a new species. Materials and Methods Adult worms were collected in Louisiana from the stomach and intestine of six naturally infected canebrake rattlesnakes, Crotalus horridus atricaudatus, and one naturally infected North American water moccasin, Agkistrodon piscivorus leucostoma. Specimens of Hexametra boddaertii (Wedl, 61), three males and three females, were acquired through the kindness of Dr. P. Araujo (Departamento de Parasitologia do Institute de Ciencias Biomedicas da Univ. Sao Paulo, Brasil). Also examined were the type specimen labelled 54

2 55 1 Figures Lateral view of left side of esophageal region of female. 2. En face view of male. 3. Dorsal view of dorsal lip of male. 4. Lateral view of female tail. 5. Lateral view of male tail showing spicule and ejaculatory duct. 6. Ventral view of male tail. Ascaris boddaertii Baird, 60 and the cotypes labelled A. rotundicaudata Linstow, 04 on deposit in the British Museum of Natural History, London (BMNH and BMNH ', , resp.), the syntypes labelled Ascaris quadrangularis Schneider, 66 from the Museum fur Naturkunde der Humboldt Universitat zu Berlin (Nr. 869), specimens of Hexametra from the U.S. National Parasite Collection (nos , 29862, 346, 42175, 42658, 42736, and 57553) and specimens of a Hexametra sp. from a Crotalus durissus collected in Costa Rica by Dr. D. Pence (Dept. of Pathology, Texas Tech Univ., Health Sciences Center, Lubbock, Texas). The specimens collected in Louisiana were fixed at room temperature in either 10% formalin, Carnoy's fixative, AFA (85 parts 50% ethanol, 10 parts form-

3 56 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY Figures Scanning electron micrograph showing en face view of female, x Scanning electron micrograph showing lateral side of dorsal lip and the extent of the dentigerous ridge. x!60. aldehyde, and 5 parts glacial acetic acid), or at 60 C in 70% ethanol. For the observation of the internal morphological features, the specimens were passed through a graded series of ethanols ending with two changes of 100% ethanol and were then cleared in Beechwood creosote or liquified phenol. En face preparations were made by mounting the excised anterior extremity in glycerin jelly. For the observation of microanatomy, tissues were processed by routine histological procedures and stained with Ehrlich's hematoxylin and eosin. Drawings were made with the aid of a camera lucida. Material examined with the scanning electron microscope was postfixed in Zenker's lixative, placed in water, frozen in liquid nitrogen, lyophilized, gold plated, and examined with a Cambridge SEM. Measurements, except on total body length, were made with the aid of an ocular micrometer. Total body length was measured by placing a hydrated worm between two panes of glass in front of a light source, tracing the worms on a piece of paper, and then measuring the length of the body outline. The small size of the pits in the shells of the eggs that were examined made it difficult to differentiate the individual pits at the circumference of the eggshell. Therefore to allow a comparison of the number of pits on the eggs, pits within an 80-Mm area on the surface were counted under 1,250 magnifications. Ten eggs from the vagina and common uterus of each of the three Brazilian females, three Costa Rican females, and three specimens from three different collections made in Louisiana were examined in this manner, as well as 10 eggs from the feces of a canebrake rattlesnake experimentally infected with the species from Louisiana. The eggs of the type material and specimens from the U.S. National Parasite Collection were measured in utero under 500 magnifications, and because they were examined through the body wall, the pits could not be clearly differentiated. The female labelled as the cotype of A. rotundicaudata and the female labelled as the type of A. quadrangularis unfortunately did not have eggs. Description Hexametra leidyi sp. n. (Figs. 1-12) GENERAL: Ascarididae Baird, 53; Angusticaecinae Skrjabin and Karokhin, 45; Hexametra Travassos,. Head with 3 distinct lips of equal size, varying in shape from square to trapezoidal with small to prominent dip in center of anterior margin (Figs. 2, 3, 7, 8). Two labial pulps enter each lip; each pulp branches anteriorly into two lobes that then branch into smaller lobuli. First major branch of each labial pulp forming cleft into which fibers from small papilla on anterior margin penetrate. Denticles present, extending along inner surface of anterior margin arid lateral edges of each lip to base (Figs. 3, 8). Esophagus 3.0% to 5.7% of total body length and without ventriculus or diverticulum (Fig. 1). Dorsal gland cell nucleus of esophagus spherical; 2 subventral gland cell nuclei spherical. Intestinal cecum occasionally present; cecum, when present, small, anteriorly directed, located on left side of esophageal-intestinal junction. Excretory pore slightly posterior to nerve ring. Excretory system with inverted "Y" shape; excretory duct extends posteriad to left lateral chord, then bifurcates when entering commissure of excretory cell. Excretory cell nucleus large, situated near posterior end of excretory cell commissure on left side. Deirids pit-like, lateral, slightly posterior to level of excretory pore. Lateral chords prominent, running length of body. Cuticle over lateral chords

4 OF WASHINGTON, VOLUME 51, NUMBER 1, JANUARY Table 1. Measurements in mm of male specimens of H. leidyi. Male paratypes Feature Holotype N X SD Range Body length Dorsal lip: Length Width at papillae Width at base No. denticles Anterior end to: Nerve ring Excretory pore Deirid Esophagus length Cecum length Body width at: Base of lips Nerve ring Excretory pore Base esophagus Maximum body width Tail length Tail width Mucron length Left spicule length % Ejaculatory duct length Right spicule length % Ejaculatory duct length Ejaculatory duct length No. left preanal papillae No. right preanal papillae No. left postanal papillae No. right postanal papillae % % % % % % %-62.0% %-60.3% with cuticular bars beginning near nerve ring and extending to level of esophageal-intestinal junction. Cuticular bars each having shape of inverted "V" or flattened "M" when seen in transverse section. MALE: Measurements as in Table 1. Testis single, beginning near midbody. Testis heavily coiled at midbody, then becoming straight and extending posteriad to about half of distance to tail where it joins vas efferens. Vas efferens continues posteriad, but then loops anteriad to about level of its junction with testis and joins seminal vesicle. Seminal vesicle extending posteriad to near tip of tail where it enters vas deferens proper that forms the ejaculatory duct. Tail flexed ventrally. Spicules equal and alate. Ratio of spicule length to ejaculatory duct length, 0.60 to Proximal portion of spicule an open cone of sclerotized cuticle; more distally becoming an elongate tubular shaft with lateral membranous wings (Figs. 5, 10). Wings short anteriad, becoming longer distally, and extending short distance beyond distal tip of sclerotized shaft. Preanal papillae unpaired with different numbers of preanal papillae in different positions on each side; on individual worms, difference between number on each side small. Adanal papillae include ventral papilla on anterior lip of anus and ventrolateral double papilla on each side (Fig. 6). Postanal papillae 3 or 4 in number, usually 2 subventral and 2 subdorsal. Subdorsal papillae occasionally fusing to form a single double papilla or rarely having only one subdorsal single papilla and three subventral papillae. Phasmids lateral, usually posterior to all postanal papillae. Tail terminating in short conical mucron. FEMALE: Measurements as in Table 2. Vulva near midbody; distance from anterior end to vulva highly correlated to total body length as shown by linear regression (df =, r2 = , y intercept = 2.165, slope = ). Vagina vera

5 58 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY Figures Transverse section of female showing the six uterine branches, x Transverse section of male showing the expanded lateral wings of the spicules at about the level of mid shaft, x 85. extending posteriad from vulva before joining vagina uterina. Vagina uterina extending to common uterus that divides posteriorly into 6 branches (Fig. 9). Each branch continuing posteriad forming seminal receptacle first and then oviduct. Oviduct reflexing anteriad to or beyond level of vulva. Ovary beginning at anterior extent of oviduct and continuing posteriad beyond end of uterine branches; here, all 6 ovaries forming tightly coiled mass. Tail straight after fixation, short, and with small mucron; mucron occasionally withdrawn into body in large specimens. Phasmids lateral and slightly anterior to terminal mucron. EGGS: Eggs spheroid to ovoid (N = 40) (Fig. 11); maximum diameter 90 fj,m (84-96 ^m, SD = 0.003), minimum diameter 81 /urn (74-89 pm, SD = 0.004). Shell approximately 5 nm thick; surface covered with fine pits (Fig. 12); 58 pits (45-73, SD = 7.8) within area of 80 nm. Taxonomic Summary DIAGNOSIS: Lips small in relation to body length; total body length 69-2 mm, dorsal lip length /urn, dorsal lip width at papillae fj,m, dorsal lip width at base /xm, Detvtigerous ridge with numerous, small denticles (141-2 per dorsal lip) extending to the base of the lip. Eggs 90 nm (84-96 /urn) by 81 /j,m (74-89 ^m); surface of egg with 58 (45-73) pits within a surface area of 80 /j-m2. SPECIMENS DEPOSITED (USNM Helminthological Collection, USDA, Beltsville, Maryland 705): holotype (male), no , allotype (female), no ; paratypes, nos HOST: Crotalus horridus atricaudatus, canebrake rattlesnake (type host). Also found in Agkistrodon piscivorus leucostoma, water moccasin. LOCALITY: Louisiana: Orleans Parish. SITE OF INFECTION: Lumen of stomach and small intestine. ETYMOLOGY: Named in honor of Dr. Joseph Leidy for his pioneering work in the field of parasitology. Remarks All H. leidyi examined differed in three major respects from H. boddaertii sensu Sprent (78) and the specimens from Costa Rica that are considered here to also be H. boddaertii. The differences were in the size of the lips, the size, number, and distribution of the denticles on the lips, and the size of the eggs and the pits on their surfaces. The lips of H. leidyi were found to be consistently smaller than on specimens of//, boddaertii of equal size. Because lip measurements are correlated with body length, the analysis of covariance test was used to compare the dimensions of the dorsal lips of H. leidyi and H. boddaertii specimens. Using this test, it was found that the differences in lip length and width at both the base of the lip and at the level of the double papillae were all significantly different in the two groups (df = 1,51; P < 0.001). The regression coefficients show that the length of the dorsal lip of//, leidyi is usually about 35 /j.m less, the width at the double papillae is usually about 60 /urn less, and the width at the base of the lips is usually about 85 ^m less than the respective dimensions on the dorsal lip of H. boddaertii.

6 OF WASHINGTON, VOLUME 51, NUMBER 1, JANUARY Table 2. Measurements in mm of female specimens of H. leidyi. Female paratypes Feature Allotype N X SD Range Body length Dorsal lip: Length Width at papillae Width at base No. denticles Anterior end to: Nerve ring Excretory pore Deirid Vulva % Body length Esophagus length Cecum length Body width at: Base of lips Nerve ring Excretory pore Base esophagus Vulva Maximum body width Tail length Tail width Mucron length Vagina length Common uterus length % 6.38 None % % %-45.7% In H. leidyi, the dentigerous ridge extends to the base of each side of the lip, whereas in H. boddaertii it extends only to the level of the anterior margin of the double papilla. Also, the denticles of H. leidyi are smaller than those of H. boddaertii. When the denticles along a distance of 55 Mm were counted from the center to the right on dorsal lips of equal size specimens, the number in H. leidyi specimens was denticles and only in H. boddaertii. However, because the size of the denticles depends largely on the size of the worm, it was concluded that it would be better to use the total number of denticles on the dorsal lip for making comparisons between species rather than the number of denticles in a given length of the dentigerous ridge. Also, the total number of denticles on the dorsal lip of each specimen was found not to be related to the size of the worm examined. When the total number of denticles was compared between the two species, H. leidyi was found to have more denticles per dorsal lip than H. boddaertii. The male specimens of H. leidyi were found to have a range of total denticles on each of their dorsal lips, whereas two of the male specimens of//, boddaertii from Brazil had only 96 and 101 denticles on each of their dorsal lips. The female specimens of //. leidyi also had more numerous denticles on each of their dorsal lips with a range of as compared with 79 and 83 for two female H. boddaertii from Brazil. The specimens of//, boddaertii from Costa Rica had denticles only on the anterior margin of their lips with the number of denticles ranging from 86 to 103. The denticles on the lips of the type specimen of H. boddaertii were not counted, but the dentigerous ridge did not extend beyond the anterior margin of the double papillae. The eggs of H. leidyi are larger than those of H. boddaertii. Sprent (80) reported egg sizes for H. boddaertii (78-88 nm by MHI) that are smaller than those reported in by Baylis (88-95 urn by pan). The present study found measurements that were in agreement with those of Sprent rather than those of Baylis. The eggs of the type specimen of H. boddaertii measured 79 Mm (74-85 Mm, N = 10, SD = 3.3) by 71 Mm (65-78 Mm, N= 10, SD = 4.3), which when combined with measurements made on eggs

7 60 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY Figures x Egg of H. leidyi. x Egg of H. leidyi showing the pits present on the eggshell. from the Brazilian and Costa Rican specimens produced measurements of 80 i*m (72-86 /itm, N = 70, SD = 3.1) by 70 Mm (65-82 urn, N = 70, SD = 3.8). The eggs of H. leidyi, on the other hand, were found to measure 90 ^m (84-96 nm) by 81 iim (74-89 yum) showing that they are on the average about 10 /urn larger in each dimension. Because the ranges overlap slightly, the means were compared using Student's /-test with pooled variances, and the difference was found to be significant (P < 0.001). It is believed that this difference in size clearly separates these two groups of eggs. Also, when the number of pits on the eggs of H. leidyi was compared to the number present on the eggs of H. boddaertii, it wa;» found that H. boddaertii had more pits within a defined area on the surface of its eggshell. The eggs from Brazilian and Costa Rican specimens each had a mean of 90 (75-114, N = 60, SD = 10.3) pits in an 80-Mm2 area of its surface. H. leidyi had a mean of about one-third less pits in the same area, i.e., 58 (45-73). Because the ranges did not overlap, it is apparent that this feature also separates H. leidyi from H. boddaertii. Only two species ofhexametra have been previously described as having denticles extending to the base of the lips. One is H. rotundicaudata (Linstow, 04) Mozgovoi, 53 from lizards of the genus Calotes in Ceylon and India, the other is H. hexauterina (Skrjabin, ) Kreis, 44 from a lance-headed viper, Bothrops sp., in Paraguay. Sprent (78) indicated in a footnote that H. rotundicaudata is possibly an immature form of H. quadricornis. However, an examination of the type specimens indicated that the number and extent of the denticles were similar to those of//, leidyi rather than those of other Hexametra species, and for this reason, it is suggested that H. rotundicaudata be considered a separate species ofhexametra occurring in lizards in Asia. Sprent (78) mentioned that the description of H. hexauterina by Skrjabin () indicated that the only difference between this species and //. boddaertii was the extent of the denticles down the lateral sides of the lips, and he synonymized it with H. boddaertii which has precedence. H. leidyi appears similar to H. hexauterina in that it has numerous denticles extending far down the sides of the lips, and eggs similar in size to those described by Skrjabin (Skrjabin measured the eggs to be 0.09 mm by 0.07 mm). In light of the description of H. leidyi, the type specimens of H. hexauterina should be reexamined, but they could not be found for study. This lack of type material along with the inadequacy of the original description accompanying the illustration showing small denticles extending to the base of a subventral lip make it impossible to determine at this time whether or not H. hexauterina is the same species as H. leidyi. Therefore, until such time as the types are found or neotypes are collected from the same host and locality, it is felt that the species H. hexauterina should be considered in a position of subjudice. Sprent (78) reported that the specimens of Hexametra from North American rattlesnakes in the U.S. National Parasite Collection all ap-

8 OF WASHINGTON, VOLUME 51, NUMBER 1, JANUARY peared to be H. boddaertii, but an examination of these worms disclosed one specimen that did not appear to be H. boddaertii sensu Sprent, 78. This was a female (USNHC 28248) collected from a western rattlesnake, Crotalus viridis. This worm had denticles extending to the base of its lips with 9 denticles on the dorsal lip. Unfortunately, this specimen is in poor condition and many other features of this worm, including the size of the eggs, could not be determined. However, the extent and number of denticles on this specimen would suggest that it probably is H. leidyi. The type specimen of H. boddaertii is from a colubrine snake, Mastigodryas boddaerti, collected in the West Indies. Sprent (78) reported that H. boddaertii occurs in both colubrid and viperid snakes in Brazil, and the worms collected by Dr. Pence indicate that it is also found in viperids in Costa Rica. The range of H. boddaertii in North America, however, is difficult to determine because all but one of the specimens from rattlesnakes in the U.S. National Parasite Collection were from zoo animals. The only worm that was not from a captive host is a single female specimen (USNHC 57553) from a "rattlesnake" collected in central Florida. H. leidyi, on the other hand, appears to be a parasite of the canebrake rattlesnake, Crotalus horridus atricaudatus, in Louisiana that is now considered by most as synonymous with the timber rattlesnake, Crotalus horridus, with a range from southwestern Maine to northern Florida west to southwest Minnesota and central Texas. Finding H. leidyi in a water moccasin appears to be a very rare occurrence, because many water moccasins have been examined in this laboratory and not found to have Hexametra although they may have other ascaridoids. The specimen in the western rattlesnake, Crotalus viridis, that had been housed in a San Diego zoo may indicate that this parasite is present in snakes farther west than Louisiana. Acknowledgments The author would like to thank Dr. M. D. Little for his numerous suggestions, criticisms, and ideas, Dr. Robert G. Yaeger for his aid in the collection and handling of the snakes from which many of the worms used in this study were obtained, Dr. J. H. Esslinger for help and suggestions in the field of zoological nomenclature, Dr. David W. Fredericksen for his aid in the preparation of the scanning electron micrographs, and Dr. J. Hughes for her aid in the statistical analyses. Thanks also to Drs. Paulo Araujo and D. Pence who supplied specimens that were used in this work. The author would also like to thank Dr. G. Hartwich of the Museum fur Naturkunde an der Humboldt Universitat zu Berlin and Dr. J. R. Lichtenfels of the U.S. National Parasite Collection for the loan of specimens. Special thanks to the staff of the helminthological collection of the British Museum of Natural History for their aid while visiting the museum. Also, the author would like to thank Dr. P. C. Beaver for reading and commenting on the manuscript. Literature Cited Araujo, P. 69. Sobre a conceituacao de helmintos, pertencentes a subfamilia Ascarinae Travassos, 13, parasitas de ofidios e lacertilios. Rev. Fac. Farm, y Bioquim., Sao Paulo Univ. 7: Baird, W. 60. Description of some new species of intestinal worms (Entozoa) in the collection of the British Museum. Proc. Zool. Soc. London 444, pt. 28: Baylis, H. A.. The types of the species of Ascaris described by Baird. Parasitology 8: On the classification of the Ascarididae. II. The Polydelphis group; with some account of the other ascarids parasitic in snakes. Parasitology 12: Kreis, H. A. 44. Beitrage zur Kenntnis parasitischer Nematoden. XI. Neue parasitischer Nematoden. Rev. Suisse Zool. 51: Skrjabin, K. I.. Contribution a 1'etude de la faune helminthologique du Paraguay. Zool. Viestnik 1: Sprent, J. F. A. 78. Ascaridoid nematodes of amphibians and reptiles: Polydelphis, Travassosascaris n.g. and Hexametra. J. Helminthol. 52: Travassos, L. P.. Esboco de uma chave geral dos nematodes parasites. Rev. de Vet. Zootech. 10:59-70.